Sperm Nuclear Shape Research Articles

Prediction of Nili-Ravi buffalo bull fertility through Fourier harmonic analysis of sperm

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0–5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0–5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (−0.0002 ± 0.07 vs. −0.266 ± 0.09), kurharm0 (−0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0–5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. −0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.

PSXII-17 Fertility prediction model for Nilli-Ravi buffalo bulls through Fourier harmonic analysis of sperm

Abstract A model to predict Nili-ravi buffalo bull fertility was developed based on Fourier harmonic analysis of sperm. Seventeen bulls with 3032 AI records were categorizes based on fertility rate (FR) as low (36.5±0.2, n = 6: SD&amp;lt; ˗1 from mean FR), medium (39.9±0.2, n = 3; SD +1 to -1 from mean FR) and high fertility (41.4±0.1, n = 8; SD &amp;gt; +1 from mean FR). Cryopreserved semen samples from these bulls were investigated for Fourier harmonic analysis of sperm nuclear shape. Hoechst-33342 and YOYO-1 fluorescent stains were used to identify live and dead sperm. Digital images were analyzed to get sperm nuclear perimeter points at different phase angles to generate Fourier functions. Mean harmonic amplitude (HA) 0 was different (P &amp;lt; 0.05) for 1700 live vs. 1294 dead sperm from the 17 bulls, thus live sperm were used for remaining analyses. The mean, variance, skewness and kurtosis values of 100 live sperm nuclei/bull were compared for HA0-5 between high (n = 6) and low (n = 6) fertility groups, considering equal number of bulls in each category. The mean HA2 (0.739±0.01 vs 0.686±0.00) and 4 (0.105±0.001 vs 0.007±0.001) were higher in high vs low fertility group respectively (P &amp;lt; 0.05). Sperm nuclear perimeter among high fertility group sperm was more elongated. There was also an increased skewness of HA0 as fertility increased (P &amp;lt; 0.05). Discriminant analysis defined a fertility model by using mean HA4, skewness HA0 and variance HA2, that resulted in 91.7% bulls into their correct fertility group upon cross-validation (canonical correlation=0.928; P &amp;lt; 0.05). Higher values of mean HA4, skewness HA0 and variance HA2 increase the chance of bulls being placed in the high fertility group. In conclusion, sperm nuclear shape in Nili-ravi buffalo bull is related to in vivo fertility. A fertility model using reported discriminant measures could be used to objectively identify Nili-ravi buffalo bulls of varying fertility.

Water buffalo bull fertility selection by sperm nuclear shape: Model development and evaluation

Dietary carotenoids have a high antioxidant capacity, so it has been hypothesised that carotenoid supplimentation will improve sperm production and quality by protecting sperm from oxidative damage. The effects of carotenoids on sperm have only been assessed in three vertebrate species, and evidence for improved sperm concentration and motility remains equivocal. One reason for this might be that in most studies there has not been an assessment of the effects of single carotenoid compounds over a range of doses. Applied research focused on developing ways to improve sperm quality could benefit the captive breeding and conservation of threatened species. The aim of the present study was to assess a dose-dependent effect of beta-carotene supplementation on sperm concentration and motility in the endangered booroolong frog (Litoria booroolongensis). Individuals were supplemented with one of four beta-carotene doses (0, 0.1, 1 and 10 mg/g) from hatching until sexual maturity (53 weeks). Sperm concentration was determined prior to activation, and percent sperm motility and sperm velocity were measured at 0, 3 and 6 h post-activation using computer-assisted sperm analysis. Unexpectedly, beta-carotene had no significant effect on sperm concentration, percent motility or velocity at any time point, providing no evidence for beneficial effects. Findings of the present study indicate there are likely to be species-specific differences in sperm production and motility that influence the risk of oxidative damage to sperm and dependence on dietary antioxidants to inhibit these detrimental effects.

Scrotal insulation and its relationship to abnormal morphology, chromatin protamination and nuclear shape of spermatozoa in Holstein-Friesian and Belgian Blue bulls

The objectives of this study were to identify the stages of spermatogenesis susceptible to elevated testicular temperature in terms of sperm motility, viability, morphology, chromatin protamination and nuclear shape. The latter two valuable parameters are not included in routine semen analysis. Scrotal insulation (SI) was applied for 48 h in 2 Holstein-Friesian (HF) and 2 Belgian Blue (BB) bulls and semen was collected at 7 d intervals along with semen collection of a non-insulated bull of each breed. Semen samples were frozen and assigned to 4 groups: period 1 (preinsulation) = −7 d and 0 d, where 0 d = initiation of SI after semen collection; period 2 = 7 d (sperm presumed in the epididymis during SI); period 3 = 14 d to 42 d (cells presumed at spermiogenesis and meiosis stages during SI); period 4 = 49 d to 63 d (cells presumed at spermatocytogenesis stage during SI). The percentages of progressively motile and viable spermatozoa as assessed by computer-assisted sperm analysis (CASA) and fluorescence microscopy, respectively were decreased whereas abnormal sperm heads, nuclear vacuoles and tail defects were increased at period 3 (P < 0.05) compared to period 1, 2 or 4 in SI bulls of both HF and BB breeds. Protamine deficient spermatozoa as observed by chromomycin A3 (CMA3) staining were more present (P < 0.05) at period 2 and 3 in both breeds compared to period 1 or 4. Sperm nuclear shape as determined by Fourier harmonic amplitude (FHA) was most affected by heat stress during period 3 (P < 0.01) and a higher response was observed in BB bulls than HF bulls. In conclusion, sperm cells at the spermiogenic and meiotic stages of development are more susceptible to heat stress. The lack of chromatin protamination is the most pertinent result of heat stress, together with subtle changes in sperm head shape, which can be detected by FHA but not by conventional semen analysis.

267 DIFFERENCES IN BULL SPERM NUCLEAR SHAPE BETWEEN FLOW-SORTED AND NON-SORTED SPERM USING FOURIER HARMONIC ANALYSIS

The objective of the study was to evaluate sperm nuclear shape in sex-sorted and non-sorted semen using Fourier Harmonic Amplitudes (FHA). Frozen semen was obtained from a single commercially available source. Mature bulls (n = 15) from the same breed with an average age of 4.42 ± 2.08 years were collected and the semen was either frozen or X-chromosome sorted using a flow cytometer and then frozen. Frozen straws were transported to the lab and analyzed for FHA. Briefly, straws were thawed and cells were incubated with 1.6 μm Hoechst 33342. Cells were then washed, fixed, dried to a slide and analyzed for nuclear head shape. Harmonic amplitudes 0 to 5 (HA0–HA5), derived from FHA, were previously shown to be an accurate, objective, and repeatable measure of sperm nuclear shape. HA0 describes the overall nuclear size of the sperm, whereas HA1 describes the anterior head, HA2 the length of the sperm along the longitudinal axis, and HA3 to 5 the distal, post-nuclear curvature of the sperm head. Each unit of semen was evaluated for motility and FHA. There was a significant decrease in motility in the sorted group (77 ± 1% v. 54 ± 3%; P &lt; 0.0001). Multivariate ANOVA showed that there were differences between the sorted and non-sorted groups in HA1 to 4 (P &lt; 0.02). Harmonic amplitude means ± SD (microns) for sorted and non-sorted treatments are as follows: HA1 (0.117 ± 0.003 v. 0.109 ± 0.003), HA2 (1.087 ± 0.005 v. 1.063 ± 0.005), HA3 (0.139 ± 0.003 v. 0.130 ± 0.003), and HA4 (0.201 ± 0.004 v. 0.191 ± 0.004), respectively. The nuclear shape of X-sorted sperm is longer and more pinched in both the anterior and posterior head. Interestingly there was no difference in HA0 (P = 0.119) indicating that the overall size of the sperm head is not affected by the sorting process. The differences in harmonic amplitudes may be due to the size and a restricted location of the X v. Y chromosome in the sperm nucleus.

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis is the product of the interaction between perinuclear microtubules and condensing chromatin. This interaction occurs during spermiogenesis and is established through the nuclear membrane. As in other cephalopod species, the perinuclear microtubules are transient structures. In the case of S. officinalis, they begin to appear in the basal area of the early spermatid and progress from there, establishing contact with the external nuclear membrane and follow a defined, but not symmetric, geometry. Thus, the microtubules accumulate preferentially in one area of the nuclear membrane which we refer to here as the “dorsal zone”. Later, the microtubules will be eliminated before the mature spermatid migrates to the epidydimis. The chromatin is condensed within the nucleus following a complex pattern, beginning as fibro-granular structures until forming fibres of approximately 45 nm diameter (patterning phases). From this stage on, an increase in the chemical basicity of DNA-interacting proteins is produced, and chromatin fibres coalesce together, being recruited to the dorsal zone of the membrane, where there is a higher density of microtubules. This last step (condensation phases) allows the chromatin fibres to be arranged parallel to the axis of the elongating nucleus, and more importantly, is deduced to cause a lateral compression of the nucleus. This lateral compression is in fact a recruitment of the ventral zone toward the dorsal zone, which brings about an important reduction in nuclear volume. The detailed observations which comprise this work complement previous studies of spermiogenesis of Sepia and other cephalopods, and will help to better understand the process of cellular morphology implicated in the evolution of sperm nuclear shape in this taxonomic group.

226 DAY LENGTH DOES NOT AFFECT LIVE SPERM NUCLEAR SHAPE IN THE STALLION

Fourier harmonic analysis (FHA) of sperm nuclei is a precise and objective method to evaluate shape of the sperm head, with the calculated harmonic amplitudes highly related to male fertility. The FHA approach has been developed for use in the bull and the boar but has not yet been applied to the stallion. Direct utilization of the previous fluorescent approaches to identify and image live sperm nuclei in the bull cannot be used in the stallion due to the increased thickness of the post-nuclear region and thin anterior region of the sperm head. An alternative approach was developed in which live and motile sperm were isolated after filtration of an ejaculate through a Sephadex G-15 column. The resulting live sperm were sonicated briefly to separate tails and heads. The heads were isolated on a 45–90 discontinous Percoll gradient, fixed with paraformaldehyde (0.2%), centrifuged onto glass slides, and dried. The slides were then stained with eosin (1%), cleared with water, and dried again; Permount was added, followed by a coverslip. Slides were imaged with phase contrast microscopy; digital images were acquired and evaluated with custom software to identify perimeter coordinates of sperm nuclei. The perimeter coordinates were next converted to Fourier harmonic amplitudes 0–5 (HA0–HA5) using trigonomic regression at 1 degree equally spaced angles. Fertility of bulls were previously reported to be most related to changes in HA0 and HA2 but no information is available on stallions. As fertility data on stallions is limited, to evaluate FHA in the equine, the day length (period of light) was increased in January from the ambient 9–10 h to 16. Semen samples from 5 light horse stallions were collected at weekly intervals for 8 weeks following the increase in light. It was hypothesized that fertility would increase for each stallion over the course of the experiment, as testosterone increases and spermatogenesis improves with increasing day length, as previously shown. Each week semen samples were evaluated for FHA live sperm nuclei, as described above. All parameter means and variations were recorded on 100 randomly selected sperm nuclei per semen sample evaluated. There was no difference relative to week 0 in the least squares mean or SD of HA0, HA1, HA2, HA3, HA4, or HA5 over the 8 weeks (P &gt; 0.05). However stallions were consistently different for all HAs (P &lt; 0.05). The overall mean HA0–HA5 � SEM were 1.973 � 0.38, 0.087 � 0.002, 0.721 � 0.021, 0.051 � 0.004, 0.208 � 0.014, and 0.031 � 0.002, respectively. Even though libido increased during the experiment, confirming the effect of light on the stallions, no affect on sperm nuclear shape or its variation was detected using FHA. Based on work in other species involving numbers of sperm inseminated, if large numbers of sperm from these stallions were inseminated in mares, we would predict no change in fertility due to season. Further research is needed to confirm this prediction.

High-resolution image cytometry of rat sperm nuclear shape, size and chromatin status: Experimental validation with the reproductive toxicant vinclozolin

Recent studies have shown that the complex inter-related processes of sperm chromatin organization and nuclear morphogenesis, both of which are important fertility determinants, may be disrupted by chemicals. A high-resolution image cytometry method has been developed, using the fluorescent dye bisbenzimide, for the measurement of 20 features of the sperm nucleus related to size, form and chromatin status in the rat. For the complete set of features measured and from a total of 150 spermatozoa assessed per sample, the overall coefficient of reproducibility was 5%. Then, an experimental validation of the method was carried out in rats chronically exposed to the antiandrogenic reproductive toxicant vinclozolin and control animals. Univariate statistics revealed significant vinclozolin-induced changes for 19 out of 20 morphometric and chromatin features. Stepwise linear discriminant analysis classified correctly 84.3% of the sperm nuclei with only four features selected. The accuracy and reproducibility of the cytometry assessment of the sperm nuclei together with the results of the experimental validation suggest this method may be a new powerful tool for use in reproductive toxicology.

Relationship of bull fertility to sperm nuclear shape.

The relationship between sperm nuclear shape and bull fertility was determined. Two groups of bulls, 3 per group, were selected. Bulls differed in fertility based on lifetime nonreturn rates. Digital images of propidium iodide-stained sperm from each bull were collected and shape-evaluated by Fourier harmonic amplitudes 0 to 5. A discriminant function (P < .05) was constructed based on harmonic amplitudes and the 2 fertility groups. When individual sperm were classified as being of high or lower fertility, the percentage of each bull's sperm placed in the high-fertility group had a linear relationship (r = .89, P < .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P < .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P < .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.

Measurement of bovine sperm nuclear shape using Fourier harmonic amplitudes.

An objective method for measuring bovine sperm nuclear shape was developed. Digital images of bovine sperm stained with propidium iodide were collected and Fourier functions used to describe the perimeters of individual sperm nuclei. Harmonic amplitudes from Fourier functions were first shown to be independent of sperm orientation during digitization. Sperm from 12 different bulls were used, and 6 harmonic amplitudes per sperm were found to adequately describe sperm nuclear shape. Based on harmonic amplitudes 0 to 5, cluster analysis was used to generate 20 different groups. Sperm within groups had similar morphologies and groups were distinguished by statistically unique shape characteristics. Harmonic amplitudes 0 to 5 can be used to distinguish previously reported abnormalities such as tapered, pyriform, macrocephalic, and microcephalic, as well as gradations in between. Furthermore, differences were detected among bull harmonic amplitude centroids (P < .05), indicating that bulls differ in mean sperm nuclear shape.

Human sperm morphometry and smoking, caffeine, and alcohol consumption

The purpose of this study is to determine whether sperm nuclear size, shape, and chromatin texture parameters are associated with lifestyle exposures including smoking, caffeine, and alcohol consumption. Eightysix healthy male volunteers (ages 18–35), recruited through newspaper advertisements, provided a semen, blood, and urine sample and completed a questionnaire concerning demographic and lifestyle exposures. Sperm nuclear size, shape, and chromatin texture parameters were measured using computerized image analysis. Results indicated no associations between the sperm nuclear morphometric parameters and age, smoking, or alcohol consumption. There was weak evidence for an association with caffeine intake. In conclusion, the lifestyle factors smoking, caffeine intake, and alcohol consumption do not appear to significantly affect sperm nuclear size, shape, or chromatin texture in this study population.

Sea urchin sperm nuclear enlargement and shape transformations are differentially regulated in vitro

To elucidate the mechanisms regulating morphogenesis of the sperm nucleus into a male pronucleus after fertilization, we have used an in vitro cell-free system derived from sea urchin eggs. Demembranated sperm nuclei were added to extracts prepared from either unfertilized eggs (unfertilized extract) or fertilized eggs (fertilized extract), and the area and shape of the sperm nuclei were measured using video-intensified fluorescence microscopy coupled with image analysis. Sperm nuclei did not decondense in buffer used to prepare the extracts, indicating decondensation factors are of maternal origin. Unfertilized extract supported sperm nuclear decondensation to a greater extent than fertilized extract. This was not due to egg activation nor different preparation methods, but was due to the fertilizing sperm nucleus binding or inactivating diffusible decondensation factors within 10 min. Sperm nuclei needed constant exposure to egg cytoplasm for greater than 30 min for decondensation to proceed. Although sperm nuclear decondensation was temperature sensitive, a thermostable (100°C) decondensing factor was present in egg cytoplasm in limited quantities. Sperm nuclear decondensation was ATP-dependent, pH-sensitive, and required kinase activity, including tyrosine kinase activity. Shape transformations in fertilized extract were insensitive to 6-dimethylaminopurine indicating the area and shape alterations are regulated differentially. In addition, alterations of shape, but not area, were sensitive to serine protease inhibitors, suggesting proteases change the sperm nuclear matrix, thereby altering shape. Sperm nuclear decondensation factors and their targets were highly conserved between sea urchin species, demonstrating a common decondensing mechanism is utilized, which may be applicable to other species. J. Exp. Zool. 277:401–416, 1997. © 1997 Wiley-Liss, Inc.

Feulgen-DNA Cytophotometry of Bovine Spermatozoa

Summary Two methods have been compared for quantitating the Feulgen-DNA in the same 240 mature bovine spermatozoa, using a fixed light beam cytophotometer. The two-wave-length method measured absorbance of single stained nuclei in a circular light beam having a diameter slightly larger than the long axis of the sperm head and eliminated the need for nuclear-size determinations. An over-all Feulgen-DNA coefficient of variability of 20.4% and a within sample coefficient of 14.6% were found. The other method measured nuclear absorbance of a monochromatic light beam with a slightly smaller diameter than the nuclear width and required an accurate nuclear volume determination related to the volume traversed by the light beam. Nuclear volume was estimated from linear measurements using three different formulae. All yielded Feulgen-DNA values with over-all coefficients of variability ranging from 12.3 to 12.9% and within sample coefficients of 6.5 to 7.3%. Two formulae gave falsely precise Feulgen-DNA values due to systematic errors in area calculation resulting from false assumptions of sperm nuclear shape. Nuclear area of each cell calculated by the van Duijn formula multiplied by its extinction value for a light beam of known area gave the best estimate of sperm nuclear Feulgen-DNA.